The present invention concerns novel methods for detecting single-stranded target nucleic acid sequences.
As used herein the term xe2x80x9cnucleic acidxe2x80x9d includes protein nucleic acid (i.e. nucleic acids in which the bases are linked by a polypeptide backbone) as well as nucleic acids (e.g. DNA and RNA) having a sugar phosphate backbone, and chemical analogues thereof.
Generally speaking, amplification techniques may be viewed as effecting the detection of a target by amplifying it. However, these typically require a large target sequence since it is to be amplified and must be unambiguously identifiable in order to reduce background noise. These techniques also amplify only the detected sequence, and are unable to provide separate distinct sequences for subsequent detection, for example sequences which will hybridise to other sequences in a detection system, or which are specifically bound by antibodies. For example, amplification techniques used to detect different targets are unable to generate a common product for all of the different targets. Amplification techniques also typically require the use of thermal cycling, thus causing the reaction to pass through many heating and cooling steps. This slows down the reaction, requires the use of expensive thermal cycler devices and of larger quantities of reagents or the use of more expensive thermostable reagents.
One example of an amplification technology is PCR (EP 0201184) in which a pair of complementary template strands are treated with a molar excess of two oligonucleotide primers so that the primers hybridize to the template strands. A polymerase enzyme is then used to extend the primers to produce strands complementary to the template strand sequences. The hybridised templates and complementary strands are then separated by heat denaturation to give two new pairs of complementary template strands. Subsequent cooling allows further primers to hybridise to the template strands. By repeating the cycle of steps, an exponential amplification of the complementary template strands is achieved. A particular disadvantage encountered with PCR is the need to cycle the reaction through many heating and cooling steps, as discussed above.
Isothermal amplification procedures are also known which similarly use a molar excess of primers but avoid the need for thermal cycling by using strand displacing polymerasc enzymes (i.e. polymerase enzymes lacking 5xe2x80x2 to 3xe2x80x2 exonuclease activity), which displace the non-template part of any double stranded DNA they encounter whilst themselves progressing along a template DNA strand to produce double stranded DNA.
Such isothermal amplification techniques are exemplified by the Strand Displacement Amplification of EP 0497272, which provides for amplification of a target nucleic acid sequence, but requires a pair of primers, at least one template strand, and the presence of at least one substituted inter-nucleotide linkages, meaning that amplification products all contain substituted deoxynucleosidetriphosphates. It also requires the use of a second set of primers in reactions.
Rolling Circle Amplification is another isothermal amplification system, requiring a circular single-stranded template. Primers hybridised to the template allow the binding and activity of strand displacing polymerases which circle around the template displacing non-template sequence, and which produce just one single stranded complementary strand containing a number of tandem repeats of the sequence complementary to the template strand. However, the unit copies within the concatamer cannot be manipulated further by processes such as extension against another template unless they have undergone additional further processing. As such they are of extremely limited use when compared to the multiple fixed-length linear products of PCR amplification.
Rolling Circle Amplification can achieve exponential amplification by including a second primer which is complementary to the concatameric strand produced by the first primer. In a cascade reaction the second primer hybridises to the concatameric strand as it is produced and the polymerase enzyme extends it (using the concatamer as template) to generate product. As the linear amplification reaction occurs further sites for hybridisation of the second primer are produced upstream of the extended product. Extension of additional second primer from these sites leads to displacement of the downstream non-template strands which in turn reveal sites for hybridisation of further first primer sequences. Thus occurs a cascade of exponential production of sites and hybridisation thereto and polymerisation of primers therefrom. A disadvantage of this method is the requirement for some pre-treatment of the target (e.g. a ligation of a synthetic nucleic acid, against the target to form a circle) and three synthetic molecules are involved in the most beneficial embodiments of the invention.
The Qxcex2 replicase amplification system relies on the target sequence to anchor a modified version of midivariant 1 (MDV1) to a solid phase, while non-anchored sequences are removed by washing. MDV1 is an RNA molecule which is the specific target for amplification by the RNA-directed RNA polymerase (replicase) of bacteriophage Qxcex2, producing up to 106 to 109 copies of MDV1 in 15 minutes. An inherent problem with the system is the need for scrupulous w ashing to remove solution phase copies of MDV1 which are, otherwise, amplified to the same extent as target bound copies, producing a high level of background xe2x80x9cnoisexe2x80x9d.
Cycling probe amplification utilises a chemically synthesised probe containing linkages, between some of its nucleotides, which (only when the molecule is rendered double stranded by hybridisation with the target) can be broken by the action of an enzyme. In a typical reaction, the target is denatured with the probe and the reaction is then held at a temperature at which intact probe molecules will hybridise to the target but at which the shorter products of enzymatic degradation will not. Thus, probe molecules hybridise, are cleaved by the enzyme, and the shorter products are displaced by the hybridisation of new probe molecules, in a cyclic reaction. The reaction is essentially linear in nature with the reaction products currently being detected by gel electrophoresis.
U.S. Pat. Nos. 5,645,987 and 5,863,732 (which are incorporated herein by reference in their entirety) disclose the enzymatic synthesis of oligonucleotides using a template strand having a priming region, intervening region and complementary region having a cutting attenuation modification. The priming region is complementary to a desired target oligonucleotide sequence. Upon hybridisation of the target sequence to the priming region, the target sequence is extended by polymerase activity to provide a complementary strand. This results in the production of a double stranded restriction endonuclease recognition sequence which is nicked by the restriction endonuclease, the template strand not being cut because of the effect of the cutting attenuation modification. Additional polymerase activity extends the 3xe2x80x2 terminus of the nicked complementary strand 5xe2x80x2 to the restriction site, displacing the complementary strand 3xe2x80x2 to the cleavage site (i.e. complementary to the complementary region of the template strand) and creating another double-stranded restriction endonuclease recognition sequence. The process can continue ad infinitum to give a linear rate of synthesis of oligonucleotides complimentary to the template strand complimentary region.
A cascade of such reactions can be employed to give non-linear rates of amplification, the displaced complimentary strand from a first reaction acting as a target oligonucleotide complimentary to the priming region of a second template strand, and so on.
WO 92/01813 and Terrance Walker, G. et al. (1992, PNAS USA, 89(1): 392-396; PMID 1309614) disclose methods of circular extension and of amplification.
No suggestion is made as to how to achieve non-linear rates of amplification using a single template strand.
The present invention overcomes the prior art disadvantages and provides new methods for synthesising oligonucleotide products and detecting single-stranded target nucleic acid sequences. Double stranded nucleic acid sequences may of course be detected by separating (melting) them to provide a pair of single stranded sequences and detecting one or both of them using the methods of the present invention.
According to the present invention there is provided a method for synthesising oligonucleotide products comprising the steps of:
(a) providing a circular nucleic acid template sequence having at least one cleavage site which, when rendered double-stranded by a complementary strand, provides a cleavage site for a cleavage agent which cleaves only said complementary strand;
(b) hybridising to said template sequence a target nucleic acid sequence to act as a primer nucleic acid sequence; and
(c) treating said template sequence and hybridised primer sequence with a strand-displacing polymerising agent and the reagents and conditions necessary to effect the action of said polymerising agent, and with said cleavage agent or agents; such that said polymerising agent produces a complementary strand hybridised to said template sequence and which is cleaved at said cleavage site or sites, such that it is subsequently displaced as a single-stranded linear nucleic acid by said polymerising agent.
Such a method may be considered a method of amplifying a template sequence to produce single-stranded sequence complementary to it.
Such a method may provide a plurality of displaced (i.e. not even hybridised in part to the template sequence) single-stranded nucleic acid sequences.
Also provided according to the present invention there is a method for detecting the presence of a target nucleic acid sequence, comprising the step of:
(a) performing a method for synthesising oligonucleotide products according to the present invention;
(b) detecting any displaced single stranded complementary strands; and
(c) correlating the results of detection step (b) with the presence of said target nucleic acid sequence.
By providing a double-strand specific cleavage site for a cleavage agent and only cleaving the complementary strand, a repeated cycling of a strand-displacing polymerising agent around the circular template produces a series of complementary strand products of equal length.
A number of primers may be hybridised to the circular template strand such that a number of complementary strands arc produced in tandem.
It may be desirable to create more than one single-stranded linear nucleic acid complementary strand product from a single circular template, and this may be achieved by providing more than one cleavage site.
Cleavage agents are well known (see below) and different cleavage sites may be cleaved by the same cleavage agent or by different cleavage agents.
A product of the method of the present invention need not include the target nucleic acid sequence and such a product could be the displaced single stranded complementary strand, for example which is detected in step (b) of the detection method.
The primer sequence may hybridise at a position on the template strand which is distant from the cleavage site, or it may hybridise at a position which is near to or which overlaps the cleavage site.
Upon cleavage, a site is presented at the 3xe2x80x2 terminus of the complementary strand which may be bound by the polymerising agent and provide a start site for polymerisation. Further cleavage allows a number of polymerising agents to proceed along the template strand at the same time.
The displaced single-stranded linear complementary strand may subsequently hybridise to a single-strand circular template sequence and act as a primer. Thus, if a molar excess of circular template sequences is provided relative to the quantity of primer nucleic acid sequence, exponential amplification of oligonucleotide synthesis may be achieved, only being limited by the quantity of circular template sequences. Such a exponential amplification using a single template sequence to provide a plurality of single stranded nucleic acid products is not suggested by the prior art, which requires e.g. a cascading series of reactions.
The circular nucleic acid may be formed from a linear nucleic acid precursor which may be circularised by any one of a number of methods. For example, the termini of the linear sequence may be hybridised to a complementary DNA strand (which might be a target molecule) and ligated (if they are adjacent), or a DNA polymerase may be used to fill any xe2x80x9cgapxe2x80x9d between the termini prior to ligation. The regions of the linear precursor, and hence the circular template, which are outside those required for hybridisation to the target can be varied to generate different amplification products. Multiple linear precursors may be directed to different sequences in the target in order to generate multiplex signals. Alternatively, different linear precursors could be directed to the same target sequence in such a way that only perfectly matched sequences would form circular molecules. Such an arrangement might find application in areas such as the detection of different mutations in a common underlying sequence.
Such an overlapping complementary linker strand may act as the primer for the polymerising agent. It may contain (in its final double-stranded form) a cleavage site, meaning that it is only when circularisation has been completed and a double stranded cleavage site produced that cleavage occurs and the polymerising agent is able to bind and produce a complementary strand. For example, the linear strand may have a 3xe2x80x2 dideoxyterminus, requiring cleavage to produce a new 3xe2x80x2-deoxy terminus to allow the polymerising agent to work. In particular by placing the primer binding site upstream of the cleavage site, this can prevent cascading polymerising agent activity before the linear nucleic acid sequence has been circularised.
The complementary strand product may be used as a primer for an additional circular template sequence, thereby providing a xe2x80x9ccascadexe2x80x9d effectxe2x80x94the product of one stage acting as a primer for the next and so on.
The additional circular template sequence may have nucleic acids added/subtracted and/or substituted to it when compared to the first template sequence on any other template sequence.
Thus oligonucleotide synthesis according to the present invention may comprise at least one additional step of providing:
(I) an additional circular nucleic acid template sequence to which will hybridise all or part of said displaced single-stranded linear nucleic acid forming a primer nucleic acid sequence;
(ii) said additional template sequence also having at least one cleavage site which, when rendered double stranded by a complementary strand, provides a cleavage site for a cleavage agent which cleaves only said complementary strand; and
(iii) treating said additional sequence and hybridised single-stranded linear nucleic acid with a strand-displacing polymerising agent and the reagents and conditions necessary to effect the action of said polymerising agent, and with said cleavage agent or agents, such that said polymerising agent produces a further complementary strand hybridised to said additional template sequence and which is cleaved at said cleavage site or sites such that it is subsequently displaced as a linear nucleic acid by said polymerising agent.
Linear template strands may also be used in such additional steps. The use of linear template strands is described in more detail below and the synthesis of oligonucleotide products may comprise at least one additional step of providing:
(I) an additional linear nucleic acid template strand to which will hybridise said displaced single-stranded linear nucleic acid forming a primer nucleic acid sequence;
(ii) said additional template strand also having at least one cleavage site which, when rendered double stranded by a complementary strand, provides a cleavage site for a cleavage agent which cleaves only said complementary strand; and
(iii) treating said additional template strand and hybridised single-stranded linear nucleic acid with a strand-displacing polymerising agent and the reagents and conditions necessary to effect the action of said polymerising agent, and with said cleavage agent or agents, such that said polymerising agent produces a further complementary strand hybridised to said additional template strand and which is cleaved at said cleavage site or sites such that it is subsequently displaced as a linear nucleic acid by said polymerising agent.
The additional circular nucleic acid template sequence or linear nucleic acid template strand may have a nucleotide or nucleotides added, subtracted and/or substituted to it when compared to the template sequence.
It is known that with existing amplification techniques such as PCR which provide a primer for subsequent extension against a sample template strand that xe2x80x9cnoisexe2x80x9d, i.e. false results, can result from unexpected hybridisation and polymerisation. Since the present invention only provides a template strand, the target nucleic acid sequence acting as a primer and being extended, it is possible to prevent such xe2x80x9cnoisexe2x80x9d resulting from the unexpected hybridisation of a linear template strand by ensuring that a linear template strand, even if subject to degradation, cannot be extended. This can be achieved by synthesising it from methyl RNA. Naturally, such problems cannot be encountered with a single circular template sequence, and this avoidance of xe2x80x9cnoisexe2x80x9d can be particularly advantageous.
A single stranded template sequence can be either partially or wholly single stranded. This can be used to allow only those portions of the template to which the primer would bind to be presented as a single strand. This protects the rest of the molecule from non-specific hybridisation, which might otherwise increase unwanted side reactions, or from hybridisation of an amplification product from an identical template which would effectively remove that product from further amplification rounds.
This protection might be provided in a number of ways. For example, a template sequence could have a loop motif downstream of the polymerising agent start site such that, under the conditions of the reaction, it would form a partial hairpin structure with the single stranded primer binding site upstream of this. On binding of the primer, and extension by the polymerase, the double stranded hairpin region would be opened by strand displacement to allow copying of the formerly protected down-stream sequence. In order for these hairpin structures to provide efficient amplification it may be necessary to include, in the loop of the hairpin, nucleotides which are modified such that they cannot serve as template for the polymerase and would terminate extension across the loop. This would prevent synthesis of a product which also contained a hairpin region, which might reform as a double strand in solution, preventing further amplification. Such termination agents might include PNA or inosine residues.
Alternatively, with both circular and linear template sequences, non-primer sequences may be bound to provide double-stranded regions, these sequences subsequently being displaced by the advancing polymerising agent. This may be advantageously used by leaving only primer regions of template strands single-stranded, thereby preventing non-specific hybridisations and subsequent amplification cycles. In the case of linear template strands this would prevent one complementary strand product from hybridising to another identical template strand and effectively removing it from further reactions. Clearly any non-primer sequence hybridised to the template strand should not be extendable by the polymerising agent. Various ways are known of rendering sequences unextendable and include incorporation of a di-deoxyribonucleotide at the 3xe2x80x2 end of the molecule. However, all oligonucleotides are subject to some degree of degradation on storage and the use of a single substituted base may not, therefore, be sufficient to maintain protection. More robust protection might be provided by using entirely modified bases such as methyl-RNA oligonucleotides.
The method of the present invention lends itself to the detection of target nucleic acid sequences which may be recalcitrant to amplification by other means (e.g. PCR) and, more particularly, may be used to achieve the optimum reaction conditions for detection of multiple sequences. This may allow the detection of sequences in multiplex reactions which would be difficult or impossible to achieve by other means due to the nature of the sequences to be amplified.
Cleavage agents, and the cleavage sites for which they are specific, are well knownxe2x80x94a cleavage site may be a restriction endonuclease site, the template strand (but not the complementary strand) being protected from endonuclease activity by chemical or other modification (e.g. phosphothioate bonds). Restriction enzymes which may be used include BstNI, AvaI, BsmAI BsoBI, BsrI, HincII (New England Biolabs). Alternatively, a cleavage site may be a complementary sequence against which is specific an enzyme which cuts only one specific strand of a DNA duplex. Examples of such enzymes are N.BstSE (Abdurashitou et al., 1996, Mol. Biol. (Mosk), 30(6): 1261-1267) and enzymes not conventionally considered to be restriction enzymes, for example the bacteriophage M13 gpII enzyme which recognises and specifically cuts one strand of a duplex at the f1 origin of replication. Cleavage agents include other than the standard class of proteinaceous enzymes, and include any agent which is capable of catalysing the specific cleavage of a complementary strand and include, for example, RNA ribozymes.
A variety of primer sequences may be used. A polymerising agent will typically be a DNA polymerasc enzyme which synthesises a complementary strand in a 5xe2x80x2 to 3xe2x80x2 direction, adding nucleotides to the 3xe2x80x2-OH terminal of the complementary strand. Thus the 3xe2x80x2 end of the primer must be hybridised to the template strand, although the 5xe2x80x2 end could, of course, overhang it. In order to be susceptible to the activity of the polymerising agent, the primer must have a free 3xe2x80x2-OH group. This can be provided for example by either a synthetic primer or by having a cleavage site within the double stranded region formed by primer hybridisation with the template strandxe2x80x94upon hybridisation the cleavage agent could act to cleave the primer strand, generating the required 3xe2x80x2-OH group. Alternatively, a cleavage agent such as a restriction enzyme could be used to cleave a double-stranded primer prior to its denaturation and subsequent binding to the template strand. Alternatively, a probe sequence may be hybridised with part of a single stranded primer sequence not possessing a 3xe2x80x2-OH group in a desired position, giving a double-stranded region and a single-stranded region. The single stranded region could then be digested using a single-strand specific nuclease enzyme, leaving a free 3xe2x80x2-OH group, and the primer and probe separated by denaturation, allowing the primer to subsequently hybridise to the template strand.
A wide range of strand-displacing polymerising agents are available and may be used in the methods of the present invention. These include Bca, Bst, Vent, Vent exoxe2x88x92; Deep Vent, Deep Vent exoxe2x88x92; 9xc2x0 North, Bacteriophage xc3x829, Klenow or Klenow exoxe2x88x92 (New England Biolabs except Bacteriophage xc3x829).
As well as simply being used to synthesise oligonucleotide products (e.g. amplify a template strand), the methods of the present invention may also be used for diagnostic purposesxe2x80x94the presence of a specific nucleic acid sequence may be detected by the use of a template strand for which the desired nucleic acid sequence is a specific primer, a sample possibly containing the desired nucleic acid sequence undergoing a detection method according to the present invention, the presence of the desired nucleic acid sequence being determined by detecting the quantity of template sequence complementary strands and correlating the quantity of template sequence complementary strands with the presence of the desired nucleic acid sequence.
Similarly, rather than providing just one template sequence, a range of template sequences may be provided, each being specific for a different primer. In this way, e.g. point mutations in a sequence may be readily detected. In such a case, the template strands could be of different lengths so that their complementary strands could be readily identified by e.g. gel electrophoresis. Alternatively, each template sequence could be provided with a unique marker sequence so that the production of complementary strands could later be determined using e.g. specific probes.
A wide range of methods are known for detecting specific single stranded nucleic acid molecules such as the displaced single stranded nucleic acids (i.e. displaced complementary strands) of the present invention. These may involve capturing the product onto a complementary solid phase oligonucleotide and detecting the structure produced, either by secondary probing or by extending one sequence against the other, including signal generating groups such as biotinylated or fluorescent nucleotides that are then detected. Alternative methods include the use of coupled fluorophore/quencher techniques, such as Molecular Beacons in which the linearisation of a loop structure bearing fluorophore and quenching moieties distances the fluorophore from the quenching moiety, allowing detectable fluorescence.
The methods of the present invention may be used to quantify the presence of any target nucleic acid sequence. The methods of the present invention may be used as diagnostic tests for the presence of a target nucleic acid sequence.